P873: CARFILZOMIB RESISTANCE IS ASSOCIATED WITH SIGNIFICANT DEREGULATION OF THE BH3 FAMILY PROTEINS
Authors: A. Schneller, A. Bolomsky, N. Zojer, M. Schreder, H. Ludwig
Journal: HemaSphere (2022)

Background: Despite the remarkable clinical activity of PIs (Proteasome inhibitors) in MM (multiple myeloma), most patients ultimately relapse and become PI-resistant. Deregulation of the apoptosis pathway has been identified as a possible cause of drug resistance in tumor cells. The intrinsic apoptosis pathway is regulated by the BCL2 protein (BH3) family. Previous studies indicated a substantial role of the BH3 family in PI resistance and in response to PI. For instance, the sensitivity to the BCL-2 inhibitor venetoclax is increased in PI resistant AMO-1 cells and a deregulation of BH3 proteins Noxa and MCL1 was noted in MM cell lines exposed to bortezomib. These data suggest that mechanisms involved in PI resistance are closely interconnected with the intrinsic apoptosis pathway orchestrated via the BH3 family, revealing novel opportunities for therapeutic intervention. Aims: Here, we analyze the interplay between BH3 protein family members in PI resistant vs. sensitive cell lines aiming to decipher survival strategies of PI resistance. Methods: MM cell lines were exposed to serially increasing doses (max. 25nM) of carfilzomib for > 1 year, leading to the outgrowth of clones with acquired carfilzomib resistance. Analysis of BH3 protein expression and sensitivity to BH3 mimetics in PI resistant cell lines were performed via western blot and viability assays, respectively. Analysis of BH3 protein dependency was done using the flow cytometry-based BH3 profiling method. Thereby, cytochrome c release was measured upon exposure to peptides specifically binding anti-apoptotic BH3 proteins, allowing to identify the relevance of distinct anti-apoptotic BH3 proteins in the cell line of interest. Apoptosis induction of the cells was analyzed by flow cytometry. Results: Viability assays indicate that all 7 tested carfilzomib resistant MM cell lines developed a cross resistance to the MCL-1 inhibitors S63845 and AZD-5991. Decreased sensitivity to the BCL-2 inhibitor venetoclax as well as to the BCL-xL inhibitor A1331852 was observed in resistant KMS12PE cells. As previously shown, these effects are linked to MDR1 upregulation in carfilzomib resistant cells and reversible by MDR-1 inhibition. BH3 profiling indicates that under naïve conditions the changes in BH3 protein dependency could be observed in 3/7 carfilzomib resistant cell lines. Specifically, resistant KMS12PE cells exhibit a decreased sensitivity to the MCL-1 binding peptide, carfilzomib resistant OPM-2 cells show an increased sensitivity towards peptides binding to BCL-xL and A1 and resistant AMO-1 cells demonstrate a reduced sensitivity a peptide binding to BCL-xL, Bcl-2 and BCL-w. Protein expression analysis of carfilzomib sensitive vs. resistant cell line variants indicate alterations of BCL2 family members (MCL-1, BCL-xL, BCL-2, BAD, BID, BAX, BAK, BIM, NOXA and PUMA) in untreated conditions as well as under carfilzomib treatment (Figure 1). Notably, carfilzomib treatment upregulated BCL-xL in 5 and BAK in 7 out of 7 carfilzomib resistant cell line variants. Image:Summary/Conclusion: Our data show significant deregulation of the BH3 protein family in carfilzomib resistant cell lines highlighting the importance of the apoptosis signaling pathway in PI resistance. This is further emphasized by the coherent deregulation of BCL-xL and BAK throughout carfilzomib exposed carfilzomib resistant cell lines when compared to their sensitive cell line variants. Moreover, BH3 profiling indicates a change in dependency on anti-apoptotic BH3 protein in carfilzomib resistant vs. sensitive MM cell lines.